TRINS: a method for gene modification by randomized tandem repeat insertions.

ABSTRACT

In nature, the evolution of new protein functions is driven not only by side-chain substitutions (point mutations), but also by backbone modifications (insertions and deletions). The current laboratory diversification methods, however, are largely limited to point mutations. Of particular interest are short insertions-by-duplication that are frequent in nature but cannot be introduced in vitro in a library format (i.e. in random locations and lengths). Here, we describe a new procedure that allows the generation of tandem repeats of random fragments of the target gene via rolling-circle amplification, and the concurrent incorporation of these repeats into the target gene. This procedure, dubbed tandem repeat insertion, or TRINS, results in a library of genes carrying insertions-by-duplication of variable lengths (3-150 bp) at random positions. This diversification pattern allows sampling of sequence space regions that are not readily accessible by other protocols. We demonstrate this method by constructing three different gene libraries, and by selecting insertion variants of TEM-1 ß-lactamase.